Figure 3.

Apc11p interacts with Ubc4 in vitro. GST pull-down assays were employed to test for interactions between Apc11p and the ubiquitin conjugating enzyme (E2) Ubc4. Purified His-tagged human Ubc4 (L, 10% of the amount added to binding reactions) was incubated in the presence of GST alone (G), GST-Apc11p (WT), or various mutant versions (41, 44, 52, 62, 80, 81, and 91, as described in Figure 2) of Apc11p expressed as GST-fusion proteins. GST-fusions and interacting proteins were isolated using glutathione-agarose beads (see MATERIALS AND METHODS), separated by SDS-PAGE, and analyzed by immunoblotting for Ubc4 (upper panel). Blots were subsequently stained with coomassie blue (lower panel) to assess the levels of GST or GST-fusion proteins isolated (arrows). The asterisk indicates a contaminating protein present in all samples.